Three major classes of opioid receptors, mu, delta and kappa, have been defined based on differences in their pharmacology, physiology and tissue distribution. When stimulated in vivo the opioid receptors activate a variety of intracellular reactions that effect calcium channels, potassium channels and adenylyl cyclase activity resulting in many of the classical effects of opiate intoxication including euphoria, analgesia and physical dependence. The molecular characterization of the opioid receptors was slow until the recent expression cloning of a mouse delta opioid receptor was reported. Based on this sequence we and others developed cloning strategies that led to the isolation of both kappa and mu opioid receptor cDNAs. The ability to express each of the opioid receptors in tissue culture permits their detailed pharmacological and physiological study. In a previous application we proposed that R21, a novel receptor we had cloned, encoded an opioid receptor based on the conservation of key amino acids and its overall homology with a mouse delta opioid receptor. Having demonstrated that R21 encodes a rat kappa opioid receptor, we are now ready to proceed with the molecular studies of this receptor. In particular we propose to extend out physiological studies to include the coupling of human and rat kappa opioid receptors to calcium and potassium channels. At the anatomical level as a first step towards evaluating the effects of chronic opiate use on kappa receptor expression we will begin to characterize its distribution in human brain material. An immunomodulatory role for opioids has been demonstrated and recently we obtained evidence that kappa opioid receptor mRNA is expressed in a mouse thymoma cell line. This provides us with an excellent opportunity to explore the effects of opioid exposure on cells of the immune system. To better understand how kappa opioid receptor expression is controlled we will characterize both the human and rat genes and in addition will attempt to identify markers that can be used in genetic linkage and association studies. Eventually the mouse kappa opioid receptor gene will be targeted and knocked out. These animals will be a valuable model system in which to evaluate the kappa receptor's role in processes ranging from synaptic transmission to behavior. Finally, we have cloned a receptor whose sequence and anatomical distribution suggest that it is a member of the opioid receptor gene family. We propose to continue the characterization of this interesting receptor using DNA sequence analysis, in vitro mutagenesis and expression studies.